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(12) United States Patent (10) Patent No.: US 7,501,286 B2 Gygiet Al

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(12) United States Patent (10) Patent No.: US 7,501,286 B2 Gygiet Al US00750 1286B2 (12) United States Patent (10) Patent No.: US 7,501,286 B2 Gygiet al. (45) Date of Patent: Mar. 10, 2009 (54) ABSOLUTE QUANTIFICATION OF Sannolo, et al., “Biomonitoring of Human Exposure to Methyl Bro PROTEINS AND MODIFIED FORMS mide by Isotope Dilution Mass Spectrometry of Peptide Adducts', THEREOF BY MULTISTAGE MASS Journal of Mass Spectrometry, J. Mass Spectrom. 34, 1028-1032 SPECTROMETRY (1999). Zhou, et al., XP-002974320“A systematic approach to the analysis of protein phosphorylation”. Nature Biotechnology, Apr. 2001, vol. 19. (75) Inventors: Steven P. Gygi, Foxboro, MA (US); pp. 375-378. Scott Anthony Gerber, Brookline, MA Vinale, et al., “Development of a Stable Isotope Dilution Assay for an (US) Accurate Quantification of Protein-Bound Ne (1-Deoxy-D-fructos 1-yl)-L-lysine Using a "C-Labeled Internal Standard”. J. Agric. (73) Assignee: President and Fellows of Harvard Food Chem, 1999, 47,5084-5092. College, Cambridge, MA (US) Desiderio, “Mass spectrometric analysis of neuropeptidergic sys tems in the human pituitary and cerebrospinal fluid'. Journal of (*) Notice: Subject to any disclaimer, the term of this Chromatography B, 731 (1999) 3-22. patent is extended or adjusted under 35 Barr, et al., “Isotope dilution-mass spectrometric quantification of U.S.C. 154(b) by 323 days. specific proteins: model application with apolipoprotein A-1’’. Clini cal Chemistry 42:10, 1676-1682 (1996). (21) Appl. No.: 10/781,047 Goshe, et al., “Phosphoprotein Isotope-Coded Affinity Tag Approach for Isolating and Quantitating Phosphopeptides in Proteome-Wide (22) Filed: Feb. 17, 2004 Analyses”. Anal. Chem. 2001, 73, 2578-2586. Gygi, et al., “Quantitative analysis of complex protein mixtures using (65) Prior Publication Data isotope-coded affinity tags'. Nature Biotechnology, vol. 17, Oct. 1999, pp. 994-955. US 2004/O229283 A1 Nov. 18, 2004 * cited by examiner (51) Int. Cl. Primary Examiner Jon P Weber GOIN 24/00 (2006.01) Assistant Examiner Bin Shen (52) U.S. Cl. ...................................................... 436/173 (74) Attorney, Agent, or Firm George W. Neuner; Gregory (58) Field of Classification Search ....................... None B. Butler, Esq.; Edwards Angell Palmer & Dodge LLP See application file for complete search history. (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS The invention provides reagents, kits and methods for detect 4,572,915 A 2f1986 Crooks ing and/or quantifying proteins in complex mixtures, such as 5,532,002 A 7/1996 Story a cell lysate. The methods can be used in high throughput 20O2/O123055 A1 9, 2002 Estell et al. assays to profile cellular proteomes. In one aspect, the inven tion provides a peptide internal standard labeled with a stable FOREIGN PATENT DOCUMENTS isotope and corresponding in amino acid sequence to the WO WOOOf 11208 3, 2000 amino acid sequence of a Subsequence of a target polypep WO WO O2/O84250 A2 10, 2002 tide. In another aspect, the peptide internal standard is labeled OTHER PUBLICATIONS at a modified amino acid residue and is used to determine the presence of, and/or quantitate the amount of a particular Gerberetal. “Direct profiling of multiple enzyme activities in human cell lysates by affinity chromatography/electrospray Ionization mass modified form of a protein. spectrometry application to clinical enzymology'. Anal. Chem. 2001, 73:1651-1657.* 18 Claims, 7 Drawing Sheets U.S. Patent Mar. 10, 2009 Sheet 1 of 7 US 7,501,286 B2 Target Protein ...RLSFVFGGTDEK. Tracer (IS) Peptide Sythesis ! HN-LSFVF*GGTDEK-COOH FIG. 1 100% o MS (M+2H)2 FG. 2 U.S. Patent Mar. 10, 2009 Sheet 2 of 7 US 7,501,286 B2 100% 100% MS/MS/MS MS/MS/MS cH MS/MS D* A. B m/Z FIG. 3B U.S. Patent Mar. 10, 2009 Sheet 3 of 7 US 7,501,286 B2 Biological sample S Homogenze ! Cell lysate(protein) Add S (e.g. 10fmol) l Trypsinze ! Cell lysate(peptides) FIG. 4A LC-MS/MSIMS Ratio (Da? D'a)=absolute quantification Time FIG. 4B U.S. Patent Mar. 10, 2009 Sheet 4 of 7 US 7,501,286 B2 U.S. Patent Mar. 10, 2009 Sheet 7 Of 7 US 7,501,286 B2 SW/SW eouepundW enee US 7,501,286 B2 1. 2 ABSOLUTE QUANTIFICATION OF gen atoms, respectively. Proteins from two cells lysates can PROTEINS AND MODIFIED FORMS be labeled independently with one or the other ICAT reagent THEREOF BY MULTISTAGE MASS at cysteinyl residues. After mixing and proteolysing the SPECTROMETRY lysates, the ICAT-labeled peptides are isolated by affinity to a biotin molecule incorporated into each ICAT reagent. ICAT GOVERNMENT GRANTS labeled peptides are analyzed by LC-MS/MS where they elute as heavy and light pairs of peptides. Quantification is At least part of the work contained in this application was performed by determining the relative expression ratio relat performed under government grant HG00041 from the ing to the amount of each ICAT-labeled peptide pair in the National Institutes of Health, U.S. Department of Health and 10 sample. Human Services. The government may have certain rights in Identification of each ICAT-labeled peptide is performed this invention. by a second stage of mass spectrometry (MS/MS) and sequence database searching. The end result is relative pro RELATED APPLICATIONS tein expression ratios on a large scale. The major drawback to 15 this technique are 1) quantification is only relative; 2) spe This application claims priority under 35 U.S.C. S 120 to cialized chemistry is required, and 3) database searches are PCT/US02/025778, filed Aug. 14, 2002, published in hindered by the presence of the large ICAT reagent molecule, English, which claims priority under 35 U.S.C. S 119(e) to and 4) relative amounts of posttranslationally modified (e.g., United States Provisional Application Ser. No. 60/312,279, phosphorylated) proteins are transparent to analysis. filed Aug. 14, 2001. SUMMARY FIELD OF THE INVENTION The present invention provides reagents, kits, and methods This invention provides methods, reagents and kits for for accurate quantification of proteins and methods for using obtaining absolute quantification of proteins and their modi 25 the same. The reagents, kits, and methods of the invention are fications directly from cell lysates. In particular, the invention useful for rapid, high throughput analysis of proteomes. provides peptide internal standards for use in high perfor In one aspect, the invention provides a method for gener mance liquid chromatography (HPLC) with online detection ating a peptide internal standard. The method comprises iden by multistage mass spectrometry (MS). tifying a real or predicted peptide digestion product of a target 30 polypeptide, determining the amino acid sequence of the BACKGROUND OF THE INVENTION peptide digestion product and synthesizing a peptide having the amino acid sequence. The peptide is labeled with a mass There is a need to provide novel methods for the quantifi altering label (e.g., by incorporating labeled amino acid resi cation of proteins and modified proteins from cell lysates. The dues during the synthesis process) and fragmented (e.g., by current standard for protein detection (quantification) is 35 multi-stage mass spectrometry). Preferably, the label is a based on immunoreactive detection (Western analysis). How stable isotope. A peptide signature diagnostic of the peptide is ever, this technique requires the availability of an appropri determined, after one or more rounds of fragmenting, and the ately specific antibody. In addition, many antibodies only signature is used to identify the presence and/or quantity of a recognize proteins in an unfolded (denatured) form, cross peptide of identical amino acid sequence in a sample. reactivity can be severely limiting, and quantification is gen 40 Preferably, a labeled peptide is provided which co-elutes erally relative. with an unlabeled peptide having the same amino acid The development of methods and instrumentation for auto sequence (i.e., a target peptide) in a chromatographic separa mated, data-dependent electrospray ionization (ESI) tandem tion procedure (e.g., Such as HPLC). mass spectrometry (MS/MS) in conjunction with microcap In one aspect, the mass-altering label is part of a peptide illary liquid chromatography (LC) and database searching 45 comprising a modification, and the peptide is fragmented to has significantly increased the sensitivity and speed of the determine a peptide signature diagnostic of Such a modified identification of gel-separated proteins. Microcapillary LC peptide. The modified residue in the peptide internal standard MS/MS has been used successfully for the large-scale iden comprises a phosphorylated residue, a glycosylated residue, tification of individual proteins directly from mixtures with an acetylated residue, a ubiquitinated residue, a ribosylated outgel electrophoretic separation (Linket al., 1999; Opiteket 50 residue, or a farnesylated residue, or another modification al., 1997). However, while these approaches dramatically found in a cellular protein. In one aspect, panels of peptide accelerate protein identification, quantities of the analyzed internal standards are generated corresponding to (i.e., diag proteins cannot be easily determined, and these methods have nostic of) different modified forms of the same protein. not been shown to Substantially alleviate the dynamic range Peptide internal standards corresponding to different pep problem also encountered by the 2DE/MS/MS approach. 55 tide Subsequences of a single target protein also can be gen Therefore, low abundance proteins in complex samples are erated to provide for redundant controls in a quantitative also difficult to analyze by the microcapillary LC/MS/MS assay. In one aspect, different peptide internal standards cor method without their prior enrichment. responding to the same target protein are generated and dif There is thus a need to provide methods for the accurate ferentially labeled (e.g., peptides are labeled at multiple sites comparison of protein expression levels between cells in two 60 to vary the amount of heavy label associated with a given different states, particularly for comparison of low abundance peptide).
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